The detection of nucleic acid is used in a wide variety of assay formats to obtain both qualitative and quantitative information about the nucleic acid content of a sample. Fluorescent dyes that complex with DNA and in turn produce a detectable signal have increased the sensitivity and quality of information gained from such experiments. However, there still remains no fluorescent dye that is capable of detecting RNA in the presence of DNA for easy and direct detection of RNA.
Currently there exists fluorescent hybridization methods for detection of RNA in the presence of DNA wherein a dye is covalently attached to a nucleic acid hybridization probe (Micklefield, et al. Nucleosides, Nucleotides and Nucleic Acids (2001) 20(4-7), 1169, Yamana, et al. Angew. Chem. Int. Ed. (2001) 40(6), 1104 and Yamana, et al. Bioconjugate Chemistry (2002) 13, 1266). However, this method is synthetically inconvenient and requires many steps to accomplish the desired results. The current invention overcomes the restrictions of the old methods by providing a simple and efficient means of selective hybridization that requires less time and less expertise to accomplish the same or better results. Herein we report novel dimer compounds that are capable of selectively detecting RNA and which can also be used for the detection of DNA.
Dimers that are known for the detection of nucleic acid include dimers of unsymmetrical cyanine dyes, (U.S. Pat. Nos. 5,410,030; 5,582,977; 6,664,047 and WO 93106482) ethidium dimers (U.S. Pat. No. 5,314,805), acridine dimers and acridine-ethidium heterodimers (U.S. Pat. No. 6,428,667 and Rye, et al. Nucleic Acids Research (1990) 19(2), 327). The following references describe DNA intercalating fluorescent dimers and their physical characteristics: Gaugain et al., Biochemistry (1978) 17:5071-5078; Gaugain et al., Biochemistry (1978) 17:5078-5088; Markovits et al., Anal. Biochemistry (1979) 94:259-269; Markovits et al. Biochemistry (1983) 22: 3231-3237; and Markovits et al., Nucl. Acids Res. (1985) 13:3773-3788. The present dimers that are described herein are not only different in structure from other dimer compounds, but differ in spectral properties, binding affinities, intracellular properties and binding kinetics.
The present invention overcomes the limitations of the known nucleic acid reporter molecules by providing reporter molecules that are capable of detecting RNA in the presence of DNA. These novel dimer compounds are attached by a linker that contains at least one aromatic, heteroaromatic, cyclic or heterocyclic moiety. The rigidity of this moiety is one factor that allows for the ability to selectively detect RNA. In addition, the present invention also provides reporter molecules that have utility for detecting DNA.